Industrial hemp cannabis cultivars and seeds with stable cannabinoid profiles

ABSTRACT

According to the invention, there is provided novel hemp  Cannabis  cultivars with THC content of 0.2% by dry weight and a unique terpene profile. This invention thus relates to the seeds of hemp  Cannabis  cultivars of the invention, to the plants of hemp  Cannabis  cultivars of the invention, to plant parts of hemp  Cannabis  cultivars of the invention, to methods for producing a  Cannabis  cultivar by crossing the hemp  Cannabis  cultivars of the invention with another  Cannabis  cultivar, and to methods for producing a  Cannabis  cultivar containing in its genetic material one or more backcross conversion traits or transgenes and to the backcross conversion  Cannabis  plants and plant parts produced by those methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. Ser. No. 15/452,925, filedMar. 8, 2017, which claims priority to previously filed provisionalapplication, U.S. Ser. No. 62/342,658, filed May 27, 2016, hereinincorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to the field of plant breeding. Inparticular, this invention relates specialty cannabis plants, cultivarsand varieties, including methods for making and using said cannabisplants and compositions derived thereof.

BACKGROUND OF THE INVENTION

Industrial hemp is legally defined in the United States as Cannabiswhich contains 0.3% or less total sample dry weight ofΔ9-Tetrahydrocannabinal (THC). THC content is normally well above the0.30% threshold in modern varieties of Cannabis. THC is one of anestimated 85 cannabinoids (a class of terpenoids) synthesized inCannabis species (El-Alfy et al., 2010, “Antidepressant-like effect ofdelta-9-tetrahydrocannabinol and other cannabinoids isolated fromCannabis sativa L”, Pharmacology Biochemistry and Behavior 95 (4):434-42).

Cannabinoids act on endogenous cannabinoid receptors located throughoutthe human body (Kreitzer and Stella, 2009, “The therapeutic potential ofnovel cannabinoid receptors”, Pharmacology & Therapeutics 122 (2):83-96). These receptors are present in humans because the human bodymanufactures a similar class of cannabinoids known as theendocannabinoids (Pertwee et al., 2010, “International Union of Basicand Clinical Pharmacology. LXXIX. Cannabinoid Receptors and TheirLigands: Beyond CB1 and CB2”, Pharmacological Reviews 62 (4): 588-631).

The demand for the medicinal properties of cannabinoids derived fromCannabis is growing. Over the last 15 years, medicinal marijuana hasgained similar regulatory ground as hemp. This is a reflection ofconsumer demand. In 2013, medical marijuana sales were estimated at 1.5billion dollars. The medicinal effects of cannabinoids on human healthcontinue to be validated as clinical research in this field expands andgains traction (Scott et al., 2014, “The Combination of Cannabidiol andΔ9-Tetrahydrocannabinol Enhances the Anticancer Effects of Radiation inan Orthotopic Murine Glioma Model”, Molecular Cancer Therapeutics 13(12): 2955-2967). The ability to create this medicine without THC ishighly desired by many patients and regulatory agencies.

Terpenes are a large class of volatile organic hydrocarbons. In plants,they function as hormones (e.g. abscisic acid), as photosyntheticpigments (e.g. carotenoids) and are involved in many other vitalphysiological processes. Secondary terpenoids (secondary metabolites)account for the majority of terpenoid molecular structural diversity.The secondary terpenoids play a major role in the plant's response toenvironmental factors such as such as pathogen and photooxidativestresses (Tholl, 2006, “Terpene synthases and the regulation, diversityand biological roles of terpene metabolism”, Current Opinion in PlantBiology 9 (3): 297-304). Apart from their functions in the plant,terpenes from hops (Humulus lupulus) such as myrcene and humulene serveas major aromatic and flavor compounds in beer. Cannabis synthesizesmany terpenes including myrcene and humulene.

Cannabis normally reproduces under a dioecious system where male(staminate) and female (pistillate) flowers develop on separate plants.Monoecious plants (containing both male and female flowers) do exist.Female floral anatomy is characterized by pistils protruding from acalyx covered with resinous glandular trichomes. The glandular trichomesof the female flower are the primary site of cannabinoid synthesis. Thefemale calyx contains ovaries and, therefore, is the site of seeddevelopment when fertilized by pollen produced by a male plant.

A vast majority of the Cannabis produced in the United States is done soby clonal propagation. Under this production scheme, meristems are cutfrom a selected plant and treated by various methods to induce rootingso that many, genetically identical progeny may be derived from theoriginal. This is primarily done because breeding Cannabis seeds whichconsistently express a particular cannabinoid profile, often elevatedfor a particular cannabinoid (e.g. THC), is generally regarded asdifficult. The simplicity of breeding varieties to be produced under aclonal reproduction system is quickly offset by the cost of clonalproduction, among other factors (Mckey et al., 2010, “The evolutionaryecology of clonally propagated domesticated plants”, New Phytologist 186(2): 318-332). There is a need in the industry for industrial hempvarieties which are reliably low in THC when produced in diverseenvironmental conditions and which express elevated levels of certainother cannabinoids. The present invention provides a Cannabis varietythat consistently and reproducibly has nearly zero THC (thus qualifyingas industrial hemp) and elevated levels of CBD.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding preferably begins with the analysis anddefinition of problems and weaknesses of the current germplasm, theestablishment of program goals, and the definition of specific breedingobjectives. The next step is preferable selection of germplasm thatpossess the traits to meet the program goals. The goal is to combine ina single variety or hybrid an improved combination of desirable traitsfrom the parental germplasm.

SUMMARY OF THE INVENTION

According to the invention, there is provided novel Hemp Cannabiscultivars, having very low levels of Δ9-Tetrahydrocanabinal (THC). Thecultivars exhibit on average less than about 0.2% THC. The cultivarsalso demonstrate elevated levels of advantageous cannabinoids such ascannabidol (CBD) and a ratio of CBD to THC of up to about 56:1. Thisinvention thus relates to the seeds of the hemp Cannabis cultivars ofthe invention, to plants of the hemp Cannabis cultivars of theinvention, to plant parts of the hemp Cannabis cultivars of theinvention, to methods for producing a Cannabis cultivar produced bycrossing the hemp Cannabis cultivars of the invention with anotherCannabis cultivar, and to methods for producing a Cannabis cultivarcontaining in its genetic material one or more backcross conversiontraits or transgenes and to the backcross conversion Cannabis plants andplant parts produced by those methods.

This invention also relates to Cannabis cultivars and plant partsderived from the hemp Cannabis cultivars of the invention, to methodsfor producing other Cannabis cultivars derived from hemp Cannabiscultivars of the invention and to the Cannabis cultivars and their partsderived by the use of those methods. This invention further relates toCannabis cultivar seeds, plants and plant parts produced by crossing thehemp Cannabis cultivars of the invention or a backcross conversion ofthe cultivars of the invention with another Cannabis cultivar.

The invention further relates to products and compositions produced orpurified from plants of the invention including the stalks, fibers,pulp, flowers, seeds, hemp and the like. Products produced form the hempcultivars of the invention can include industrial textiles, buildingmaterials, foods, personal hygiene products such as soap, lotions, balmsand the like, animal bedding, industrial products such as paints, inks,solvents and lubricants, consumer textiles, animal feed, etc. Theinvention also relates to use of the Cannabis plants, plant partsextracts and the like as a flavoring or aromatic component in maltbeverages and the like.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of the presentinvention, the following definitions are provided:

The invention provides cannabis plants. As used herein, the term “plant”refers to plants in the genus of Cannabis and plants derived thereof.Such as cannabis plants produced via asexual reproduction and via seedproduction.

The invention provides plant parts. As used herein, the term “plantpart” refers to any part of a plant including but not limited to theembryo, shoot, root, stem, seed, stipule, leaf, petal, flower bud,flower, ovule, bract, trichome, branch, petiole, internode, bark,pubescence, tiller, rhizome, frond, blade, ovule, pollen, stamen, andthe like. The two main parts of plants grown in some sort of media, suchas soil or vermiculite, are often referred to as the “above-ground”part, also often referred to as the “shoots”, and the “below-ground”part, also often referred to as the “roots”. Plant part may also includecertain extracts such as kief or hash which includes cannabis trichomesor glands.

The term “a” or “an” refers to one or more of that entity; for example,“a gene” refers to one or more genes or at least one gene. As such, theterms “a” (or “an”), “one or more” and “at least one” are usedinterchangeably herein. In addition, reference to “an element” by theindefinite article “a” or “an” does not exclude the possibility thatmore than one of the elements is present, unless the context clearlyrequires that there is one and only one of the elements.

As used herein, a “landrace” refers to a local variety of a domesticatedplant species which has developed largely by natural processes, byadaptation to the natural and cultural environment in which it lives.The development of a landrace may also involve some selection by humansbut it differs from a formal breed which has been selectively breddeliberately to conform to a particular formal, purebred standard oftraits.

The invention provides plant cultivars. As used herein, the term“cultivar” means a group of similar plants that by structural featuresand performance (i.e., morphological and physiological characteristics)can be identified from other varieties within the same species.Furthermore, the term “cultivar” variously refers to a variety, strainor race of plant that has been produced by horticultural or agronomictechniques and is not normally found in wild populations. The termscultivar, variety, strain and race are often used interchangeably byplant breeders, agronomists and farmers.

The term “variety” as used herein has identical meaning to thecorresponding definition in the International Convention for theProtection of New Varieties of Plants (UPOV treaty), of Dec. 2, 1961, asRevised at Geneva on Nov. 10, 1972, on Oct. 23, 1978, and on Mar. 19,1991. Thus, “variety” means a plant grouping within a single botanicaltaxon of the lowest known rank, which grouping, irrespective of whetherthe conditions for the grant of a breeder's right are fully met, can bei) defined by the expression of the characteristics resulting from agiven genotype or combination of genotypes, ii) distinguished from anyother plant grouping by the expression of at least one of the saidcharacteristics and iii) considered as a unit with regard to itssuitability for being propagated unchanged.

As used herein, the term “inbreeding” refers to the production ofoffspring via the mating between relatives. The plants resulting fromthe inbreeding process are referred to herein as “inbred plants” or“inbreds.”

The term LOQ as used herein refers to the limit of quantitation for GasChromatography (GC) and High Performance Liquid Chromatographymeasurements.

The term secondary metabolites as used herein refers to organiccompounds that are not directly involved in the normal growth,development, or reproduction of an organism. In other words, loss ofsecondary metabolites does not result in immediate death of saidorganism.

The term single allele converted plant as used herein refers to thoseplants which are developed by a plant breeding technique calledbackcrossing wherein essentially all of the desired morphological andphysiological characteristics of an inbred are recovered in addition tothe single allele transferred into the inbred via the backcrossingtechnique.

The invention provides samples. As used herein, the term “sample”includes a sample from a plant, a plant part, a plant cell, or from atransmission vector, or a soil, water or air sample.

The invention provides progeny. As used herein, the term “progeny”refers to any plant resulting from a vegetative or sexual reproductionfrom one or more parent plants or descendants thereof. For instance aprogeny plant may be obtained by cloning or selfing of a parent plant orby crossing two parent plants and include selfings as well as the F1 orF2 or still further generations. An F1 is a first-generation progenyproduced from parents at least one of which is used for the first timeas donor of a trait, while offspring of second generation (F2) orsubsequent generations (F3, F4, etc.) are specimens produced fromselfings of F1's F2's etc. An F1 may thus be (and usually is) a hybridresulting from a cross between two true breeding parents (true-breedingis homozygous for a trait), while an F2 may be (and usually is) anprogeny resulting from self-pollination of said F1 hybrids.

The invention provides methods for crossing a first plant with a secondplant. As used herein, the term “cross”, “crossing”, “cross pollination”or “cross-breeding” refer to the process by which the pollen of oneflower on one plant is applied (artificially or naturally) to the ovule(stigma) of a flower on another plant. Backcrossing is a process inwhich a breeder repeatedly crosses hybrid progeny, for example a firstgeneration hybrid (F1), back to one of the parents of the hybridprogeny. Backcrossing can be used to introduce one or more single locusconversions from one genetic background into another.

The term backcrossing is a process in which a breeder crosses progenyback to one of the parents one or more times, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

The invention provides donor plants and recipient plants. As usedherein, “donor plants” refer to the parents of a variety which containsthe gene or trait of interest which is desired to be introduced into asecond variety (e.g., “recipient plants”).

In some embodiments, the present invention provides methods forobtaining plant genotypes comprising recombinant genes. As used herein,the term “genotype” refers to the genetic makeup of an individual cell,cell culture, tissue, organism (e.g., a plant), or group of organisms.

In some embodiments, the present invention provides homozygotes. As usedherein, the term “homozygote” refers to an individual cell or planthaving the same alleles at one or more loci.

In some embodiments, the present invention provides homozygous plants.As used herein, the term “homozygous” refers to the presence ofidentical alleles at one or more loci in homologous chromosomalsegments.

In some embodiments, the present invention provides hemizygotes. As usedherein, the term “hemizygotes” or “hemizygous” refers to a cell, tissue,organism or plant in which a gene is present only once in a genotype, asa gene in a haploid cell or organism, a sex-linked gene in theheterogametic sex, or a gene in a segment of chromosome in a diploidcell or organism where its partner segment has been deleted.

In some embodiments, the present invention provides heterozygotes. Asused herein, the terms “heterozygote” and “heterozygous” refer to adiploid or polyploid individual cell or plant having different alleles(forms of a given gene) present at least at one locus. In someembodiments, the cell or organism is heterozygous for the gene ofinterest which is under control of the synthetic regulatory element.

The invention provides methods for obtaining plant lines comprisingrecombinant genes. As used herein, the term “line” is used broadly toinclude, but is not limited to, a group of plants vegetativelypropagated from a single parent plant, via tissue culture techniques ora group of inbred plants which are genetically very similar due todescent from a common parent(s). A plant is said to “belong” to aparticular line if it (a) is a primary transformant (T0) plantregenerated from material of that line; (b) has a pedigree comprised ofa T0 plant of that line; or (c) is genetically very similar due tocommon ancestry (e.g., via inbreeding or selfing). In this context, theterm “pedigree” denotes the lineage of a plant, e.g. in terms of thesexual crosses affected such that a gene or a combination of genes, inheterozygous (hemizygous) or homozygous condition, imparts a desiredtrait to the plant.

The invention provides open-pollinated populations. As used herein, theterms “open-pollinated population” or “open-pollinated variety” refer toplants normally capable of at least some cross-fertilization, selectedto a standard, that may show variation but that also have one or moregenotypic or phenotypic characteristics by which the population or thevariety can be differentiated from others. A hybrid, which has nobarriers to cross-pollination, is an open-pollinated population or anopen-pollinated variety.

The invention provides self-pollination populations. As used herein, theterm “self-crossing”, “self pollinated” or “self-pollination” means thepollen of one flower on one plant is applied (artificially or naturally)to the ovule (stigma) of the same or a different flower on the sameplant.

The invention provides ovules and pollens of plants. As used herein whendiscussing plants, the term “ovule” refers to the female gametophyte,whereas the term “pollen” means the male gametophyte.

The invention provides plant tissue. As used herein, the term “planttissue” refers to any part of a plant. Examples of plant organs include,but are not limited to the leaf, stem, root, tuber, seed, branch,pubescence, nodule, leaf axil, flower, pollen, stamen, pistil, petal,peduncle, stalk, stigma, style, bract, fruit, trunk, carpel, sepal,anther, ovule, pedicel, needle, cone, rhizome, stolon, shoot, pericarp,endosperm, placenta, berry, stamen, and leaf sheath.

The invention provides methods for obtaining plants comprisingrecombinant genes through transformation. As used herein, the term“transformation” refers to the transfer of nucleic acid (i.e., anucleotide polymer) into a cell. As used herein, the term “genetictransformation” refers to the transfer and incorporation of DNA,especially recombinant DNA, into a cell.

The invention provides transformants comprising recombinant genes. Asused herein, the term “transformant” refers to a cell, tissue ororganism that has undergone transformation. The original transformant isdesignated as “T0” or “T₀.” Selfing the T0 produces a first transformedgeneration designated as “T1” or “T₁.” In some embodiments, the presentinvention provides organisms with recombinant genes. As used herein, an“organism” refers any life form that has genetic material comprisingnucleic acids including, but not limited to, prokaryotes, eukaryotes,and viruses. Organisms of the present invention include, for example,plants, animals, fungi, bacteria, and viruses, and cells and partsthereof.

As used herein, the term “female” refers to Cannabis plants carryingonly pistillate flowers and devoid of pollen. The term “bud” refers toCannabis female floral tissue collected prior to seed harvest from theapical meristems. The term “chaff” refers to Cannabis bud tissuecollected after threshing and separation of physiologically mature seedfrom the bud. The term “male” refers to Cannabis plants carrying onlystaminate flowers producing pollen.

Cannabis

Cannabis has long been used for drug and industrial purposes includingfiber, seed and seed oils, and for medicinal purposes. Industrial hempfiber products are made from Cannabis plants selected to produce anabundance of stalk tissue from which fiber is created.

Cannabis plants produce a unique family of terpeno-phenolic compoundscalled cannabinoids. Cannabinoids, terpenoids, and other compounds aresecreted by glandular trichomes that occur most abundantly on the floralcalyxes and bracts of female plants. As a drug it usually comes in theform of dried flower buds (marijuana), resin (hashish), or variousextracts collectively known as hashish oil. There are at least 483identifiable chemical constituents known to exist in the cannabis plant(Rudolf Brenneisen, 2007, Chemistry and Analysis of Phytocannabinoids(cannabinoids produced by cannabis) and other Cannabis Constituents, InMarijuana and the Cannabinoids, ElSohly, ed.; incorporated herein byreference) and at least 85 different cannabinoids have been isolatedfrom the plant (El-Alfy, Abir T, et al., 2010, “Antidepressant-likeeffect of delta-9-tetrahydrocannabinol and other cannabinoids isolatedfrom Cannabis sativa L”, Pharmacology Biochemistry and Behavior 95 (4):434-42; incorporated herein by reference). The two cannabinoids usuallyproduced in greatest abundance are cannabidiol (CBD) and/orΔ-9-tetrahydrocannabinol (THC). THC is psychoactive while CBD is not.See, ElSohly, ed. (Marijuana and the Cannabinoids, Humana Press Inc.,321 papers, 2007), which is incorporated herein by reference in itsentirety, for a detailed description and literature review on thecannabinoids found in marijuana.

Cannabinoids are the most studied group of secondary metabolites inCannabis. Most exist in two forms, as acids and in neutral(decarboxylated) forms. The acid form is designated by an “A” at the endof its acronym (i.e. THCA). The phytocannabinoids are synthesized in theplant as acid forms, and while some decarboxylation does occur in theplant, it increases significantly post-harvest and the kinetics increaseat high temperatures. (Sanchez and Verpoorte 2008). The biologicallyactive forms for human consumption are the neutral forms.Decarboxylation is usually achieved by thorough drying of the plantmaterial followed by heating it, often by either combustion,vaporization, or heating or baking in an oven. Unless otherwise noted,references to cannabinoids in a plant include both the acidic anddecarboxylated versions (e.g., CBD and CBDA).

The cannabinoids in cannabis plants include, but are not limited to, Δ 9Tetrahydrocannabinol (.Δ9-THC), A. 8-Tetrahydrocannabinol (Δ8-THC),Cannabichromene (CBC), Cannabicyclol (CBL), Cannabidiol (CBD),Cannabielsoin (CBE), Cannabigerol (CBG), Cannabinidiol (CBND),Cannabinol (CBN), Cannabitriol (CBT), and their propyl homologs,including, but are not limited to cannabidivarin (CBDV),Δ.9-Tetrahydrocannabivarin (THCV), cannabichromevarin (CBCV), andcannabigerovarin (CBGV). See Holley et al. (Constituents of Cannabissativa L. XI Cannabidiol and cannabichromene in samples of knowngeographical origin, J. Pharm. Sci. 64:892-894, 1975) and De Zeeuw etal. (Cannabinoids with a propyl side chain in Cannabis, Occurrence andchromatographic behavior, Science 175:778-779), each of which is hereinincorporated by reference in its entirety for all purposes. Non-THCcannabinoids can be collectively referred to as “CBs”, wherein CBs canbe one of THCV, CBDV, CBGV, CBCV, CBD, CBC, CBE, CBG, CBN, CBND, and CBTcannabinoids.

Cannabis Chemistry

Cannabinoids are a class of diverse chemical compounds that activatecannabinoid receptors of the human endocannabinoid physiological system.Cannabinoids produced by plants are called phytocannabinoids, a.k.a.,natural cannabinoids, herbal cannabinoids, and classical cannabinoids.At least 85 different cannabinoids have been isolated from the cannabisplants (El-Alfy et al., 2010, “Antidepressant-like effect ofdelta-9-tetrahydrocannabinol and other cannabinoids isolated fromCannabis sativa L”, Pharmacology Biochemistry and Behavior 95 (4):434-42; Brenneisen, supra). Typical cannabinoids isolated from cannabisplants include, but are not limited to, Tetrahydrocannabinol (THC),Cannabidiol (CBD), CBG (Cannabigerol), CBC (Cannabichromene), CBL(Cannabicyclol), CBV (Cannabivarin), THCV (Tetrahydrocannabivarin), CBDV(Cannabidivarin), CBCV (Cannabichromevarin), CBGV (Cannabigerovarin),and CBGM (Cannabigerol Monomethyl Ether). In the Cannabis plant,cannabinoids are synthesized and accumulated as cannabinoid acids (e.g.,cannabidiolic acid (CBDA)). When the herbal product is dried, stored, orheated, the acids decarboxylize gradually or completely into neutralforms (e.g., CBDA→CBD).

Known as delta-9-tetrahydrocannabinol (Δ9-THC), THC is the principalpsychoactive constituent (or cannabinoid) of the cannabis plant. Theinitially synthesized and accumulated form in plant is THC acid (THCA).

THC has mild to moderate analgesic effects, and Cannabis can be used totreat pain by altering transmitter release on dorsal root ganglion ofthe spinal cord and in the periaqueductal gray. Other effects includerelaxation, alteration of visual, auditory, and olfactory senses,fatigue, and appetite stimulation. THC has marked antiemetic properties,and may also reduce aggression in certain subjects (Hoaken (2003).“Drugs of abuse and the elicitation of human aggressive behavior”.Addictive Behaviors 28: 1533-1554).

The pharmacological actions of THC result from its partial agonistactivity at the cannabinoid receptor CB1, located mainly in the centralnervous system, and the CB2 receptor, mainly expressed in cells of theimmune system (Pertwee, 2006, “The pharmacology of cannabinoid receptorsand their ligands: An overview”. International Journal of Obesity 30:S13-S18.) The psychoactive effects of THC are primarily mediated by itsactivation of CB1G-protein coupled receptors, which result in a decreasein the concentration of the second messenger molecule cAMP throughinhibition of adenylate cyclase (Elphick et al., 2001, “The neurobiologyand evolution of cannabinoid signaling”. Philosophical Transactions ofthe Royal Society B: Biological Sciences 356 (1407): 381-408.) It isalso suggested that THC has an anticholinesterase action which mayimplicate it as a potential treatment for Alzheimer's and Myasthenia(Eubanks et al., 2006, “A Molecular Link Between the Active Component ofMarijuana and Alzheimer's Disease Pathology”. Molecular Pharmaceutics 3(6): 773-7.)

In the cannabis plant, THC occurs mainly as tetrahydrocannabinolic acid(THCA, 2-COOH-THC). Geranyl pyrophosphate and olivetolic acid react,catalyzed by an enzyme to produce cannabigerolic acid, which is cyclizedby the enzyme THC acid synthase to give THCA. Over time, or when heated,THCA is decarboxylated to produce THC. The pathway for THCA biosynthesisis similar to that which produces the bitter acid humulone in hops. SeeFellermeier et al., (1998, “Prenylation of olivetolate by a hemptransferase yields cannabigerolic acid, the precursor oftetrahydrocannabinol”. FEBS Letters 427 (2): 283-5); de Meijer et al. I,II, III, and IV (I: 2003, Genetics, 163:335-346; II: 2005, Euphytica,145:189-198; III: 2009, Euphytica, 165:293-311; and IV: 2009, Euphytica,168:95-112.)

CBD is a cannabinoid found in cannabis. Cannabidiol has displayedsedative effects in animal tests (Pickens, 1981, “Sedative activity ofcannabis in relation to its delta′-trans-tetrahydrocannabinol andcannabidiol content”. Br. J. Pharmacol. 72 (4): 649-56). Some research,however, indicates that CBD can increase alertness, and attenuate thememory-impairing effect of THC. (Nicholson et al., June 2004, “Effect ofDelta-9-tetrahydrocannabinol and cannabidiol on nocturnal sleep andearly-morning behavior in young adults” J Clin Psychopharmacol 24 (3):305-13; Morgan et al., 2010, “Impact of cannabidiol on the acute memoryand psychotomimetic effects of smoked cannabis: naturalistic study, TheBritish Journal of Psychiatry, 197:258-290). It may decrease the rate ofTHC clearance from the body, perhaps by interfering with the metabolismof THC in the liver. Medically, it has been shown to relieve convulsion,inflammation, anxiety, and nausea, as well as inhibit cancer cell growth(Mechoulam, et al., 2007, “Cannabidiol—recent advances”. Chemistry &Biodiversity 4 (8): 1678-1692.) Recent studies have shown cannabidiol tobe as effective as atypical antipsychotics in treating schizophrenia(Zuardi et al., 2006, “Cannabidiol, a Cannabis sativa constituent, as anantipsychotic drug” Braz. J. Med. Biol. Res. 39 (4): 421-429.). Studieshave also shown that it may relieve symptoms of dystonia (Consroe, 1986,“Open label evaluation of cannabidiol in dystonic movement disorders”.The International journal of neuroscience 30 (4): 277-282). CBD reducesgrowth of aggressive human breast cancer cells in vitro and reducestheir invasiveness (McAllister et al., 2007, “Cannabidiol as a novelinhibitor of Id-1 gene expression in aggressive breast cancer cells”.Mol. Cancer. Ther. 6 (11): 2921-7.)

Cannabidiol has shown to decrease activity of the limbic system (deSouza Crippa et al., “Effects of Cannabidiol (CBD) on Regional CerebralBlood Flow”. Neuropsychopharmacology 29 (2): 417-426.) and to increasesocial interaction which is often decreased by THC (Malon et al.,“Cannabidiol reverses the reduction in social interaction produced bylow dose Δ9-tetrahydrocannabinol in rats”. Pharmacology Biochemistry andBehavior 93 (2): 91-96.) It's also shown that Cannabidiol reducesanxiety in social anxiety disorder (Bergamaschi et al., 2003,“Cannabidiol Reduces the Anxiety Induced by Simulated Public Speaking inTreatment-Naive Social Phobia Patients”. Neuropsychopharmacology 36 (6):1219-1226). Cannabidiol has also been shown as being effective intreating an often drug-induced set of neurological movement disordersknown as dystonia (Snider et al., 1985, “Beneficial and Adverse Effectsof Cannabidiol in a Parkinson Patient with Sinemet-Induced DystonicDyskinesia”. Neurology, (Suppl 1): 201.) Morgan et al. reported thatstrains of cannabis which contained higher concentrations of Cannabidioldid not produce short-term memory impairment vs. strains which containedsimilar concentrations of THC (2010, “Impact of cannabidiol on the acutememory and psychotomimetic effects of smoked cannabis:naturalisticstudy:naturalistic study [corrected.” ]. British Journal of Psychiatry197 (4): 285-90.)

Cannabidiol acts as an indirect antagonist of cannabinoid agonists. CBDis an antagonist at the putative new cannabinoid receptor, GPR55.Cannabidiol has also been shown to act as a 5-HT1A receptor agonist, anaction which is involved in its antidepressant, anxiolytic, andneuroprotective effects. Cannabidiol is also an allosteric modulator atthe Mu and Delta opioid receptor sites.

Cannabis produces CBD-carboxylic acid through the same metabolic pathwayas THC, until the last step, where CBDA synthase performs catalysisinstead of THCA synthase. See Marks et al. (2009, “Identification ofcandidate genes affecting Δ9-tetrahydrocannabinol biosynthesis inCannabis sativa”. Journal of Experimental Botany 60 (13): 3715-3726.)and Meijer et al. I, II, III, and IV. Non-limiting examples of CBDvariants include:

CBG is a non-psychoactive cannabinoid found in the Cannabis genus ofplants. Cannabigerol is found in higher concentrations in hemp ratherthan in varieties of Cannabis cultivated for high THC content and theircorresponding psychoactive properties. Cannabigerol has been found toact as a high affinity α-2-adrenergic receptor agonist, moderateaffinity 5-HT1A receptor antagonist, and low affinity CB.sub.1 receptorantagonist. It also binds to the CB₂ receptor. Cannabigerol has beenshown to relieve intraocular pressure, which may be of benefit in thetreatment of glaucoma (Craig et al. 1984, “Intraocular pressure, oculartoxicity and neurotoxicity after administration of cannabinol orcannabigerol” Experimental eye research 39 (3):251-259). Cannabigerolhas also been shown to reduce depression in animal models (U.S. patentapplication Ser. No. 11/760,364). Non-limiting examples of CBG variantsinclude:

CBN is a psychoactive substance cannabinoid found in Cannabis sativa andCannabis indica/afghanica. It is also a metabolite oftetrahydrocannabinol (THC). CBN acts as a weak agonist of the CB1 andCB2 receptors, with lower affinity in comparison to THC.

CBC bears structural similarity to the other natural cannabinoids,including tetrahydrocannabinol, tetrahydrocannabivarin, cannabidiol, andcannabinol, among others. Evidence has suggested that it may play a rolein the anti-inflammatory and anti-viral effects of cannabis, and maycontribute to the overall analgesic effects of cannabis. Non-limitingexamples of CBC variants include:

Cannabivarin, also known as cannabivarol or CBV, is a non-psychoactivecannabinoid found in minor amounts in the hemp plant Cannabis sativa. Itis an analog of cannabinol (CBN) with the side chain shortened by twomethylene bridges (—CH2-). CBV is an oxidation product oftetrahydrocannabivarin (THCV, THV).

CBDV is a non-psychoactive cannabinoid found in Cannabis. It is ahomolog of cannabidiol (CBD), with the side-chain shortened by twomethylene bridges (CH2 units). Cannabidivarin has been found reduce thenumber and severity of seizures in animal models (U.S. patentapplication Ser. No. 13/075,873). Plants with relatively high levels ofCBDV have been reported in feral populations of C. indica (=C. sativassp. indica var. kafiristanica) from northwest India, and in hashishfrom Nepal.

THCV, or THV is a homologue of tetrahydrocannabinol (THC) having apropyl (3-carbon) side chain. This terpeno-phenolic compound is foundnaturally in Cannabis, sometimes in significant amounts. Plants withelevated levels of propyl cannabinoids (including THCV) have been foundin populations of Cannabis sativa L. ssp. indica (=Cannabis indica Lam.)from China, India, Nepal, Thailand, Afghanistan, and Pakistan, as wellas southern and western Africa. THCV has been shown to be a CB1 receptorantagonist, i.e. it blocks the effects of THC. Tetrahydrocannabinol hasbeen shown to increase metabolism, help weight loss and lowercholesterol in animal models (U.S. patent application Ser. No.11/667,860).

Cannabicyclol (CBL) is a non-psychotomimetic cannabinoid found in theCannabis species. CBL is a degradative product like cannabinol. Lightconverts cannabichromene to CBL. Non-limiting examples of CBL variantsinclude:

Terpenes and Terpenoids in Cannabis Plants

Terpenes are a large and diverse class of organic compounds, produced byCannabis plants. They are often strong smelling and thus may have had aprotective function. Terpenes are derived biosynthetically from units ofisoprene, which has the molecular formula C₅.H₈. The basic molecularformulae of terpenes are multiples of that, (C₅H₈)_(n) where n is thenumber of linked isoprene units. The isoprene units may be linkedtogether “head to tail” to form linear chains or they may be arranged toform rings. Non-limiting examples of terpenes include Hemiterpenes,Monoterpenes, Sesquiterpenes, Diterpenes, Sesterterpenes, Triterpenes,Sesquarterpenes, Tetraterpenes, Polyterpenes, and Norisoprenoids.

Terpenoids, a.k.a. isoprenoids, are a large and diverse class ofnaturally occurring organic chemicals similar to terpenes, derived fromfive-carbon isoprene units assembled and modified in thousands of ways.Most are multicyclic structures that differ from one another not only infunctional groups but also in their basic carbon skeletons. Plantterpenoids are used extensively for their aromatic qualities. They playa role in traditional herbal remedies and are under investigation forantibacterial, antineoplastic, and other pharmaceutical functions. Theterpene Linalool for example, has been found to have anti-convulsantproperties (Elisabetsky et al., Phytomedicine, May 6(2):107-13 1999).Well-known terpenoids include citral, menthol, camphor, salvinorin A inthe plant Salvia divinorum, and the cannabinoids found in Cannabis.Non-limiting examples of terpenoids include, Hemiterpenoids, 1 isopreneunit (5 carbons); Monoterpenoids, 2 isoprene units (10 C);Sesquiterpenoids, 3 isoprene units (15 C); Diterpenoids, 4 isopreneunits (20 C) (e.g. ginkgolides); Sesterterpenoids, 5 isoprene units (25C); Triterpenoids, 6 isoprene units (30 C) (e.g. sterols);Tetraterpenoids, 8 isoprene units (40 C) (e.g. carotenoids); andPolyterpenoid with a larger number of isoprene units.

In addition to cannabinoids, Cannabis also produces over 120 differentterpenes (Russo 2011, Taming THC: potential cannabis synergy andphytocannabinoid-terpenoid entourage effects, British Journal ofPharmacology, 163:1344-1364). Within the context and verbiage of thisdocument the terms ‘terpenoid’ and ‘terpene’ are used interchangeably.Cannabinoids are odorless, so terpenoids are responsible for the uniqueodor of Cannabis, and each variety has a slightly different profile thatcan potentially be used as a tool for identification of differentvarieties or geographical origins of samples (Hillig 2004. “Achemotaxonomic analysis of terpenoid variation in Cannabis” BiochemSystem and Ecology 875-891). It also provides a unique and complexorganoleptic profile for each variety that is appreciated by both noviceusers and connoisseurs. In addition to many circulatory and musculareffects, some terpenes interact with neurological receptors. A fewterpenes produced by Cannabis plants also bind weakly to Cannabinoidreceptors. Some terpenes can alter the permeability of cell membranesand allow in either more or less THC, while other terpenes can affectserotonin and dopamine chemistry as neurotransmitters. Terpenoids arelipophilic, and can interact with lipid membranes, ion channels, avariety of different receptors (including both G-protein coupled odorantand neurotransmitter receptors), and enzymes. Some are capable ofabsorption through human skin and passing the blood brain barrier.

Generally speaking, terpenes are considered to be pharmacologicallyrelevant when present in concentrations of at least 0.05% in plantmaterial (Hazekamp and Fischedick 2010. “Metabolic fingerprinting ofCannabis sativa L., cannabinoids and terpenoids for chemotaxonomic anddrug standardization purposes” Phytochemistry 2058-73; Russo 2011,Taming THC: potential cannabis synergy and phytocannabinoid-terpenoidentourage effects, British Journal of Pharmacology, 163:1344-1364).Thus, although there are an estimated 120 different terpenes, only a feware produced at high enough levels to be detectable, and fewer stillwhich are able to reach pharmacologically relevant levels.

A Cannabis terpene profile is includes the absolute and relative valuesof the 25 of the most measured terpenes disclosed herein, including butnot limited to: terpinolene, alpha phellandrene, beta ocimene, carene,limonene, gamma terpinene, alpha pinene, alpha terpinene, beta pinene,camphene, alpha terpineol, alpha humulene, beta caryophyllene, linalool,caryophyllene oxide, and myrcene. Both experts and consumers believethat there are biochemical and phenomenological differences betweendifferent varieties of cannabis, which are attributed to their uniquerelative cannabinoid and terpenoid ratios. This is known as theentourage effect and is generally considered to result in plantsproviding advantages over only using the natural products that areisolated from them (Russo 2011, Taming THC: potential cannabis synergyand phytocannabinoid-terpenoid entourage effects, British Journal ofPharmacology, 163:1344-1364).

Terpenoids can be extracted from the plant material by steamdistillation (giving you essential oil) or vaporization, however theyield varies greatly by plant tissue, type of extraction, age ofmaterial, and other variables (McPartland and Russo 2001 “Cannabis andCannabis Extracts: Greater Than the Sum of Their Parts?” HayworthPress). Typically, the yield of terpenoids in Cannabis is less than 1%by weight on analysis; however, it is thought that they may comprise upto 10% of the trichome content. Monoterpenoids are especially volatile,thus decreasing their yield relative to sesquiterpenoids (Russo 2011,Taming THC: potential cannabis synergy and phytocannabinoid-terpenoidentourage effects, British Journal of Pharmacology, 163:1344-1364).

D-Limonene is a monoterpenoid that is widely distributed in nature andoften associated with citrus. It has strong anxiolytic properties inboth mice and humans, apparently increasing serotonin and dopamine inmouse brain. D-limonene has potent anti-depressant activity wheninhaled. It is also under investigation for a variety of differentcancer treatments, with some focus on its hepatic metabolite, perillicacid. There is evidence for activity in the treatment of dermatophytesand gastro-oesophageal reflux, as well as having general radicalscavenging properties (Russo 2011, Taming THC: potential cannabissynergy and phytocannabinoid-terpenoid entourage of British Journal ofPharmacology, 163:1344-1364).

β-Myrcene is a monoterpenoid also found in cannabis, and has a varietyof pharmacological effects. It is often associated with a sweet fruitlike taste. It reduces inflammation, aids sleep, and blocks hepaticcarcinogenesis, as well as acting as an analgesic and muscle relaxant inmice. When βmyrcene is combined with Δ9-THC it could intensify thesedative effects of Δ9-THC, causing the well-known “couch-lock” effectthat some Cannabis users experience (Russo 2011, Taming THC: potentialcannabis synergy and phytocannabinoid-terpenoid entourage effects,British Journal of Pharmacology, 163:1344-1364).

D-Linalool is a monoterpenoid with very well-known anxiolytic effects.It is often associated with lavender, and frequented used inaromatherapy for its sedative impact. It acts as a local anaesthetic andhelps to prevent scarring from burns, is anti-nociceptive in mice, andshows antiglutamatergic and anticonvulsant activity. Its effects onglutamate and GABA neurotransmitter systems are credited with giving itits sedative, anxiolytic, and anticonvulsant activities (Russo 2011,Taming THC: potential cannabis synergy and phytocannabinoid-terpenoidentourage effects, British Journal of Pharmacology, 163:1344-1364).

α-Pinene is a monoterpene common in nature, also with a plethora ofeffects on mammals and humans. It acts as an acetylcholinesteraseinhibitor which aids memory and counteracts the short-term memory lossassociated with Δ9-THC intoxication, is an effective antibiotic agent,and shows some activity against MRSA. In addition, α-pinene is abronchodilator in humans and has anti-inflammatory properties via theprostaglandin E-1 pathway (Russo 2011, Taming THC: potential cannabissynergy and phytocannabinoid-terpenoid entourage effects, BritishJournal of Pharmacology, 163:1344-1364).

β-Caryophyllene is often the most predominant sesquiterpenoid incannabis. It is less volatile than the monoterpenoids, thus it is foundin higher concentrations in material that has been processed by heat toaid in decarboxylation. It is very interesting in that it is a selectivefull agonist at the CB2 receptor, which makes it the onlyphytocannabinoid found outside the cannabis genus. In addition, it hasanti-inflammatory and gastric cytoprotective properties, and may evenhave anti-malarial activity.

Caryophyllene oxide is another sesquiterpenoid found in cannabis, whichhas antifungal and anti-platelet aggregation properties. As an aside, itis also the molecule that drug-sniffing dogs are trained to find (Russo2011, Taming THC: potential cannabis synergy andphytocannabinoid-terpenoid entourage effects, British Journal ofPharmacology, 163:1344-1364).

Nerolidol is a sesquiterpene that is often found in citrus peels thatexhibits a range of interesting properties. It acts as a sedative,inhibits fungal growth, and has potent anti-malarial and antileishmanialactivity. It also alleviated colon adenomas in rats (Russo 2011, TamingTHC: potential cannabis synergy and phytocannabinoid-terpenoid entourageeffects, British Journal of Pharmacology, 163:1344-1364). Phytol is aditerpene often found in cannabis extracts. It is a degradation productof chlorophyll and tocopherol. It increases GABA expression andtherefore could be responsible the relaxing effects of green tea andwild lettuce. It also prevents vitamin-A induced teratogenesis byblocking the conversion of retinol to its dangerous metabolite,all-trans-retinoic acid (Russo 2011, Taming THC: potential cannabissynergy and phytocannabinoid-terpenoid entourage effects, BritishJournal of Pharmacology, 163:1344-1364).

Cultivars of the Invention

Cannabis NWG331

Cannabis NWG331 is a hemp Cannabis cultivar with less than 0.2% ofΔ9-Tetrahydrocannabinal (THC). The plants exhibit elevated levels ofcannabidiol (CBD) and a ratio of CBD/THC of up to about 83:1. Thecultivar produces plants with an average cannabidiol (CBD) content ofmore than 1.07% based upon total dry weight of the plant. It wasgenerated from pedigree breeding with bulk and singleseed descentselections methods, and is genetically uniform and stable.

Cannabis NWG331 is a dioecious cultivar with male and female flowersthat flowers at 58 days after planting. The plant height averages 170 cmto 190 cm and is medium height for a Cannabis cultivar. The plant hasmedium branching. The middle third of the plant is characterized bymedium stem internode length, green stem color, green leaf color, mediumleaf intensity, and medium leaf size. The cultivar has medium depth andwidth of stem grooves. Leaf anthocyanin coloration and male floweranthocyanin collation is absent. Hairs on the calyx are present but notin high density or length. Seed size is a thousand kernel weight of 13.5grams and seed shape is spherical.

Table 1 below shows a typical profile of terpene content (ppm) forNWG331 as determined by head-space Gas Chromatography (Hs-GC) with flameionization in female bud tissue.

TABLE 1 a-pinene 499.46 camphene 7.55 sabinene 6.87 myrcene 209.55b-pinene 120.12 a-phellandrene 12.43 3-carene 11.19 a-terpenine 11.74cineole ocimene-1 7.11 limonene 9.42 p-cymene ocimene-2 446.16eucalyptol 17.38 g-terpenine 10.00 terpinolene 99.04 linalool 19.01fenchone 4.88 isopulegol 13.40 borneol 4.78 terpineol 18.83 citronellol6.49 geraniol citral-1 pulegone citral-2 8.20 b-caryophyllene 1163.76humulene 309.15 nerolidol-1 38.20 nerolidol-2 29.75 guaiol 10.39caryophyllene oxide 19.81 a-bisabolol 65.61Table 2 shows a typical cannabinoid content estimate as determined byHigh-performance liquid chromatography (% dry wt) in female bud tissueharvested from NWG331.

TABLE 2 THC % THC-A % THC-total CBD % CBD-A % CBD-total CBN % CBG %CBD:THC 0.01 0.09 0.09 0.03 2.50 2.54 0.00 0.00 28.32

Cannabis NWG452

Cannabis NWG452 is a hemp Cannabis cultivar with less than 0.2% ofΔ9-Tetrahydrocannabinal (THC). The plants exhibits elevated levelscannabidiol (CBD) and a ratio of CBD/THC of up to about 83:1. Thecultivar produces plants with an average cannabidiol (CBD) content ofmore than 1.07% based upon total dry weight of the plant. It wasgenerated from pedigree breeding with bulk and single-seed descentselections methods, and is genetically uniform and stable.

Table 3 below shows a typical profile of terpene content (ppm) forNWG452 as determined by head-space Gas Chromatography (Hs-GC) with flameionization in female bud tissue.

TABLE 3 a-pinene 269.10 camphene 6.28 sabinene 3.83 myrcene 311.61b-pinene 81.61 a-phellandrene 10.58 3-carene 6.24 a-terpenine 7.84cineole ocimene-1 12.72 limonene 11.03 p-cymene ocimene-2 281.22eucalyptol 26.07 g-terpenine 5.86 terpinolene 48.13 linalool 12.63fenchone 4.34 isopulegol borneol 10.72 terpineol 15.79 citronellol 5.91geraniol citral-1 pulegone 2.04 citral-2 8.67 b-caryophyllene 1079.27humulene 300.55 nerolidol-1 19.55 nerolidol-2 62.85 guaiol 11.23caryophyllene oxide 23.46 a-bisabolol 30.89

The terpene profile of NWG452, shows that its myrcene content is 50%higher relative to NWG331.

Table 4 shows a typical cannabinoid content estimate as determined byHigh-performance liquid chromatography (% dry wt) in female bud tissueharvested from NWG452

TABLE 4 THC % THC-A % THC-total CBD % CBD-A % CBD-total CBN % CBG %CBD:THC 0.00 0.10 0.10 0.01 2.82 2.83 0.00 0.00 28.0

Further Embodiments of the Invention

This invention is also directed to methods for producing a Cannabisplant by crossing a first parent Cannabis plant with a second parentCannabis plant, wherein the first parent Cannabis plant or second parentCannabis plant is the Cannabis plant from cultivar NWG331 or NWG452.Further, both the first parent Cannabis plant and second parent Cannabisplant may be from cultivar NWG331 or NWG452. Therefore, any methodsusing hemp Cannabis cultivars NWG331 or NWG452 are part of thisinvention, such as selfing, backcrosses, hybrid breeding, and crosses topopulations. Plants produced using hemp Cannabis cultivars of theinvention as at least one parent are within the scope of this invention.

In one aspect of the invention, methods for developing novel plant typesare presented. In one embodiment the specific type of breeding method ispedigree selection, where both single plant selection and mass selectionpractices are employed. Pedigree selection, also known as the “Vilmorinsystem of selection,” is described in Fehr, Walter; Principles ofCultivar Development, Volume I, Macmillan Publishing Co., which ishereby incorporated by reference.

In one embodiment, the pedigree method of breeding is practiced whereselection is first practiced among F₂ plants. In the next season, themost desirable F₃ lines are first identified, and then desirable F₃plants within each line are selected. The following season and in allsubsequent generations of inbreeding, the most desirable families areidentified first, then desirable lines within the selected families arechosen, and finally desirable plants within selected lines are harvestedindividually. A family refers to lines that were derived from plantsselected from the same progeny row the preceding generation.

Using this pedigree method, two parents may be crossed using anemasculated female and a pollen donor (male) to produce F₁ offspring.The F₁ may be self-pollinated to produce a segregating F₂ generation.Individual plants may then be selected which represent the desiredphenotype in each generation (F₃, F₄, F₅, etc.) until the traits arehomozygous or fixed within a breeding population.

In addition to crossing, selection may be used to identify and isolatenew Cannabis lines. In Cannabis selection, Cannabis seeds are planted,the plants are grown and single plant selections are made of plants withdesired characteristics. Seed from the single plant selections may beharvested, separated from seeds of the other plants in the field andre-planted. The plants from the selected seed may be monitored todetermine if they exhibit the desired characteristics of the originallyselected line. Selection work is preferably continued over multiplegenerations to increase the uniformity of the new line.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.Backcross breeding may be used to transfer one or a few favorable genesfor a highly heritable trait into a desirable cultivar. This approachhas been used extensively for breeding disease-resistant cultivars.Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes. The use ofrecurrent selection in self-pollinating crops depends on the ease ofpollination, the frequency of successful hybrids from each pollination,and the number of hybrid offspring from each successful cross.

Each breeding program may include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard, theoverall value of the advanced breeding lines, and the number ofsuccessful cultivars produced per unit of input (e.g., per year, perdollar expended, etc.).

In one embodiment, promising advanced breeding lines are thoroughlytested and compared to appropriate standards in environmentsrepresentative of the commercial target area(s). The best lines arecandidates for new commercial cultivars; those still deficient in a fewtraits are used as parents to produce new populations for furtherselection.

These processes, which lead to the final step of marketing anddistribution, usually take several years from the time the first crossor selection is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of Cannabis plant breeding is to develop new, unique andsuperior Cannabis cultivars. In one embodiment, the breeder initiallyselects and crosses two or more parental lines, followed by repeatedselfing and selection, producing many new genetic combinations. Thebreeder can theoretically generate billions of different geneticcombinations via crossing, selfing and mutations. Preferably, each yearthe plant breeder selects the germplasm to advance to the nextgeneration. This germplasm may be grown under different geographical,climatic and soil conditions, and further selections are then made,during and at the end of the growing season.

In a preferred embodiment, the development of commercial Cannabiscultivars requires the development of Cannabis varieties, the crossingof these varieties, and the evaluation of the crosses. Pedigree breedingand recurrent selection breeding methods may be used to developcultivars from breeding populations. Breeding programs may combinedesirable traits from two or more varieties or various broad-basedsources into breeding pools from which cultivars are developed byselfing and selection of desired phenotypes. The new cultivars may becrossed with other varieties and the hybrids from these crosses areevaluated to determine which have commercial potential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents which possess favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁'s or by intercrossing two F₁'s (sib mating). Selection of the bestindividuals is usually begun in the F₂ population; then, beginning inthe F₃, the best individuals in the best families are usually selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F₄ generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (e.g., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals may be identified or created byintercrossing several different parents. The best plants may be selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. Preferably, the selected plants are intercrossed toproduce a new population in which further cycles of selection arecontinued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror line that is the recurrent parent. The source of the trait to betransferred is called the donor parent. The resulting plant is expectedto have the attributes of the recurrent parent (e.g., cultivar) and thedesirable trait transferred from the donor parent. After the initialcross, individuals possessing the phenotype of the donor parent may beselected and repeatedly crossed (backcrossed) to the recurrent parent.The resulting plant is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

The single-seed descent procedure refers to planting a segregatingpopulation, harvesting a sample of one seed per plant, and using theone-seed sample to plant the next generation. When the population hasbeen advanced from the F₂ to the desired level of inbreeding, the plantsfrom which lines are derived will each trace to different F₂individuals. The number of plants in a population declines eachgeneration due to failure of some seeds to germinate or some plants toproduce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In addition to phenotypic observations, the genotype of a plant can alsobe examined. There are many laboratory-based techniques available forthe analysis, comparison and characterization of plant genotype; amongthese are Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length polymorphisms (AFLPs), Simple Sequence Repeats(SSRs—which are also referred to as Microsatellites), and SingleNucleotide Polymorphisms (SNPs).

Isozyme Electrophoresis and RFLPs have been widely used to determinegenetic composition. Shoemaker and Olsen, (Molecular Linkage Map ofSoybean (Glycine max) p 6.131-6.138 in S. J. O'Brien (ed) Genetic Maps:Locus Maps of Complex Genomes, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1993)) developed a molecular genetic linkage mapthat consisted of 25 linkage groups with about 365 RFLP, 11 RAPD, threeclassical markers and four isozyme loci. See also, Shoemaker, R. C.,RFLP Map of Soybean, p 299-309, in Phillips, R. L. and Vasil, I. K.,eds. DNA-Based Markers in Plants, Kluwer Academic Press, Dordrecht, theNetherlands (1994).

SSR technology is currently the most efficient and practical markertechnology; more marker loci can be routinely used and more alleles permarker locus can be found using SSRs in comparison to RFLPs. Forexample, Diwan and Cregan described a highly polymorphic microsatellitelocus in soybean with as many as 26 alleles. (Diwan, N. and Cregan, P.B., Theor. Appl. Genet. 95:22-225, 1997.) SNPs may also be used toidentify the unique genetic composition of the invention and progenyvarieties retaining that unique genetic composition. Various molecularmarker techniques may be used in combination to enhance overallresolution.

Molecular markers, which include markers identified through the use oftechniques such as Isozyme Electrophoresis, RFLPs, RAPDs, AP-PCR, DAF,SCARs, AFLPs, SSRs, and SNPs, may be used in plant breeding. One use ofmolecular markers is Quantitative Trait Loci (QTL) mapping. QTL mappingis the identification of markers which are closely linked to allelesthat have measurable effects on a quantitative trait. Selection in thebreeding process is based upon the accumulation of markers linked to thepositive effecting alleles and/or the elimination of the markers linkedto the negative effecting alleles from the plant's genome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select toward the genome of the recurrent parent and against themarkers of the donor parent. This procedure attempts to minimize theamount of genome from the donor parent that remains in the selectedplants. It can also be used to reduce the number of crosses back to therecurrent parent needed in a backcrossing program. The use of molecularmarkers in the selection process is often called genetic marker enhancedselection or marker-assisted selection. Molecular markers may also beused to identify and exclude certain sources of germplasm as parentalvarieties or ancestors of a plant by providing a means of trackinggenetic profiles through crosses.

Mutation breeding is another method of introducing new traits intoCannabis varieties. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation (such as X-rays, Gamma rays,neutrons, Beta radiation, or ultraviolet radiation), chemical mutagens(such as base analogs like 5-bromo-uracil), antibiotics, alkylatingagents (such as sulfur mustards, nitrogen mustards, epoxides,ethyleneamines, sulfates, sulfonates, sulfones, or lactones), azide,hydroxylamine, nitrous acid or acridines. Once a desired trait isobserved through mutagenesis the trait may then be incorporated intoexisting germplasm by traditional breeding techniques. Details ofmutation breeding can be found in Principles of Cultivar Development byFehr, Macmillan Publishing Company, 1993.

The production of double haploids can also be used for the developmentof homozygous varieties in a breeding program. Double haploids areproduced by the doubling of a set of chromosomes from a heterozygousplant to produce a completely homozygous individual. For example, seeWan et al., Theor. Appl. Genet., 77:889-892, 1989.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Principles of Plant Breeding John Wiley and Son, pp.115-161, 1960; Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr,1987; “Carrots and Related Vegetable Umbelliferae”, Rubatzky, V. E., etal., 1999).

Cannabis is an important and valuable crop. Thus, a continuing goal ofCannabis plant breeders is to develop stable, high yielding Cannabiscultivars that are agronomically sound. To accomplish this goal, theCannabis breeder preferably selects and develops Cannabis plants withtraits that result in superior cultivars.

This invention also is directed to methods for producing a Cannabiscultivar plant by crossing a first parent Cannabis plant with a secondparent Cannabis plant wherein either the first or second parent Cannabisplant is a Cannabis plant of the line NWG331 or NWG452. Further, bothfirst and second parent Cannabis plants can come from the cultivarNWG331 or NWG452. Still further, this invention also is directed tomethods for producing a cultivar NWG331 or NWG452-derived Cannabis plantby crossing cultivar NWG331 or NWG452 with a second Cannabis plant andgrowing the progeny seed, and repeating the crossing and growing stepswith the cultivar NWG331 or NWG452-derived plant from 0 to 7 times.Thus, any such methods using the cultivar NWG331 or NWG452 are part ofthis invention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using cultivar NWG331 orNWG452 as a parent are within the scope of this invention, includingplants derived from cultivar NWG331 or NWG452. Advantageously, thecultivar is used in crosses with other, different, cultivars to producefirst generation (F₁) Cannabis seeds and plants with superiorcharacteristics.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which Cannabis plants can beregenerated, plant calli, plant clumps and plant cells that are intactin plants or parts of plants, such as embryos, pollen, ovules, flowers,seeds, roots, anthers, and the like.

As is well known in the art, tissue culture of Cannabis can be used forthe in vitro regeneration of a Cannabis plant. Tissue culture of varioustissues of Cannabis and regeneration of plants therefrom is well knownand widely published. For example, reference may be had to Teng et al.,HortScience. 1992, 27: 9, 1030-1032 Teng et al., HortScience. 1993, 28:6, 669-1671, Zhang et al., Journal of Genetics and Breeding. 1992, 46:3, 287-290, Webb et al., Plant Cell Tissue and Organ Culture. 1994, 38:1, 77-79, Curtis et al., Journal of Experimental Botany. 1994, 45: 279,1441-1449, Nagata et al., Journal for the American Society forHorticultural Science. 2000, 125: 6, 669-672. It is clear from theliterature that the state of the art is such that these methods ofobtaining plants are, and were, “conventional” in the sense that theyare routinely used and have a very high rate of success. Thus, anotheraspect of this invention is to provide cells which upon growth anddifferentiation produce Cannabis plants having the physiological andmorphological characteristics of variety NWG331 or NWG452.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively astransgenes. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed line.

Plant transformation preferably involves the construction of anexpression vector that will function in plant cells. Such a vector maycomprise DNA comprising a gene under control of or operatively linked toa regulatory element (for example, a promoter). The expression vectormay contain one or more such operably linked gene/regulatory elementcombinations. The vector(s) may be in the form of a plasmid, and can beused alone or in combination with other plasmids, to provide transformedCannabis plants, using transformation methods as described below toincorporate transgenes into the genetic material of the Cannabisplant(s).

Expression Vectors for Cannabis Transformation Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalsconfers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci.U.S.A., 80:4803 (1983). Another commonly used selectable marker gene isthe hygromycin phosphotransferase gene which confers resistance to theantibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299(1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford et al., Plant Physiol.86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab etal., Plant Mol. Biol. 14:197 (1990<Hille et al., Plant Mol. Biol. 7:171(1986). Other selectable marker genes confer resistance to herbicidessuch as glyphosate, glufosinate or broxynil. Comai et al., Nature317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618 (1990) andStalker et al., Science 242:419-423 (1988).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67(1987), Shah et al., Science 233:478 (1986), Charest et al., Plant CellRep. 8:643 (1990).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include beta.-glucuronidase (GUS),.beta.-galaetesidase, luciferase and chloramphenicol, acetyltransferase.Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teen et al., EMBOJ. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131(1987), DeBlock et al., EMBO J. 3:1681 (1984).

Recently, in vivo methods for visualizing GUS activity that do notrequire destruction of plant tissue have been made available. MolecularProbes publication 2908, Imagene Green™, p. 1-4 (1993) and Naleway etal., J. Cell Biol. 115:151a (1991). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie et al., Science 263:802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Promoters

Genes included in expression vectors preferably are driven by nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, promoter includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may affect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive promoter” is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression inCannabis. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in Cannabis. With an inducible promoter therate of transcription increases in response to an inducing agent. Anyinducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Meft et al., PNAS 90:4567-4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz etal., Mol. Gen. Genetics 227:229-237 (1991). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

B. Constitutive Promoters

A constitutive promoter may be operably linked to a gene for expressionin Cannabis or the constitutive promoter may operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in Cannabis.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810-812 (1985) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genetics 231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3):291-300 (1992)). The ALS promoter, Xbal/Ncol fragment 5′ to the Brassicanapus ALS3 structural gene (or a nucleotide sequence similarity to saidXbal/Ncol fragment), represents a particularly useful constitutivepromoter. See PCT application WO96/30530.

C. Tissue-Specific or Tissue Preferred Promoters

A tissue-specific promoter may be operably linked to a gene forexpression in Cannabis. Optionally, the tissue-specific promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in Cannabis. Plantstransformed with a gene of interest operably linked to a tissue-specificpromoter produce the protein product of the transgene exclusively, orpreferentially, in a specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6:217-224 (1993).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Close, P. S.,Master's Thesis, Iowa State University (1993), Knox, C., et al.,Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley, Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol.91:124-129 (1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuokaet al., Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell.Biol. 108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon,et al., A short amino acid sequence able to specify nuclear location,Cell 39:499-509 (1984), Steifel, et al., Expression of a maize cell wallhydroxyproline-rich glycoprotein gene in early leaf and root vasculardifferentiation, Plant Cell 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants that areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is Cannabis. In anotherpreferred embodiment, the biomass of interest is seed. For transgenicplants that show higher levels of expression, a genetic map can begenerated, primarily via conventional RFLP, PCR and SSR analysis, whichidentifies the approximate chromosomal location of the integrated DNAmolecule. For exemplary methodologies in this regard, see Glick andThompson, Methods in Plant Molecular Biology and Biotechnology CRCPress, Boca Raton 269:284 (1993). Map information concerning chromosomallocation is useful for proprietary protection of a subject transgenicplant. If unauthorized propagation is undertaken and crosses made withother germplasm, the map of the integration region can be compared tosimilar maps for suspect plants, to determine if the latter have acommon parentage with the subject plant. Map comparisons may involvehybridizations, RFLP, PCR, SSR and sequencing, all of which areconventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant line can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. Tomato encodes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

B. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

C. A lectin. See, for example, the disclosure by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See PCT applicationUS93/06487, the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani etal., Biosci. Biotoch. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

I. An enzyme responsible for a hyper accumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung Cannabis calmodulin cDNA clones, andGriess et al., Plant Physiol. 104:1467 (1994), who provide thenucleotide sequence of a maize calmodulin cDNA clone.

L. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of tachyolesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

M. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-β, lytic peptide analog to rendertransgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

O. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. CfTaylor et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

P. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Q. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb at al., Bio/Technology10:1436 (1992). The cloning and characterization of a gene which encodesa Cannabis endopolygalacturonase-inhibiting protein is described byToubart et al., Plant J. 2:367 (1992).

R. A development-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bioi/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

S. A Cannabis mosaic potyvirus (LMV) coat protein gene introduced intoLactuca sativa in order to increase its resistance to LMV infection. SeeDinant et al., Molecular Breeding. 1997, 3: 1, 75-86.

2. Genes that Confer Resistance to an Herbicide, for Example:

A. A herbicide that inhibits the growing point or meristem, such as animidazalinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance impaired by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase, PAT and Streptomyces hygroscopicusphosphinothricin-acetyl transferase PAT bar genes), and pyridinoxy orphenoxy propionic acids and cycloshexones (ACCase inhibitor-encodinggenes). See, for example, U.S. Pat. No. 4,940,835 to Shah, et al., whichdiscloses the nucleotide sequence of a form of EPSP which can conferglyphosate resistance. A DNA molecule encoding a mutant aroA gene can beobtained under ATCC accession number 39256, and the nucleotide sequenceof the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. Seealso Umaballava-Mobapathie in Transgenic Research. 1999, 8: 1, 33-44that discloses Lactuca sativa resistant to glufosinate. European patentapplication No. 0 333 033 to Kumada at al., and U.S. Pat. No. 4,975,374to Goodman et al., disclose nucleotide sequences of glutamine synthetasegenes which confer resistance to herbicides such as L-phosphinothricin.The nucleotide sequence of a phosphinothricin-acetyl-transferase gene isprovided in European application No. 0 242 246 to Leemans et al.,DeGreef et al., Bio/Technology 7:61 (1989), describe the production oftransgenic plants that express chimeric bar genes coding forphosphinothricin acetyl transferase activity. Exemplary of genesconferring resistance to phenoxy propionic acids and cycloshexones, suchas sethoxydim and haloxyfop are the Accl-S1, Accl-S2 and Accl-S3 genesdescribed by Marshall et al., Theor. Appl. Genet. 83:435 (1992).

C. A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

D. Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants. See Hattori et al., Mol. Gen.Genet. 246:419, 1995. Other genes that confer tolerance to herbicidesinclude a gene encoding a chimeric protein of rat cytochrome P4507A1 andyeast NADPH-cytochrome P450 oxidoreductase (Shiota et al., PlantPhysiol., 106:17, 1994), genes for glutathione reductase and superoxidedismutase (Aono et al., Plant Cell Physiol. 36:1687, 1995), and genesfor various phosphotransferases (Datta et al., Plant Mol. Biol. 20:619,1992).

E. Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306; 6,282,837;5,767,373; and international publication WO 01/12825.

3. Genes that Confer or Contribute to a Value-Added Trait, Such as:

A. Increased iron content of the Cannabis, for example by transforming aplant with a soybean ferritin gene as described in Goto et al., ActaHorticulturae. 2000, 521, 101-109. Parallel to the improved iron contentenhanced growth of transgenic Cannabis s was also observed in earlydevelopment stages.

B. Decreased nitrate content of leaves, for example by transforming aCannabis with a gene coding for a nitrate reductase. See for exampleCurtis et al., Plant Cell Report. 1999, 18: 11, 889-896.

C. Increased sweetness of the Cannabis by transferring a gene coding formonellin that elicits a flavor sweeter than sugar on a molar basis. SeePenarrubia et al., Biotechnology. 1992, 10: 5, 561-564.

D. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci.USA 89:2625 (1992).

E. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteriol. 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet. 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene), Penet al., Bio/Technology 10:292 (1992) (production of transgenic plantsthat express Bacillus licheniformis .alpha.-amylase), Elliot et al.,Plant Molec. Biol. 21:515 (1993) (nucleotide sequences of tomatoinvertase genes), Sogaard et al., J. Biol. Chem. 268:22480 (1993)(site-directed mutagenesis of barley .alpha.-amylase gene), and Fisheret al., Plant Physiol. 102:1045 (1993) (maize endosperm starch branchingenzyme II).

4. Genes that Control Male-Sterility

A. Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT. See international publication WO 01/29237.

B. Introduction of various stamen-specific promoters. See internationalpublications WO 92/13956 and WO 92/13957.

C. Introduction of the barnase and the barstar genes. See Paul et al.,Plant Mol. Biol. 19:611-622, 1992).

Methods for Cannabis Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

A. Agrobacterium-Mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch et al., Science 227:1229 (1985). Curtis et al., Journal ofExperimental Botany. 1994, 45: 279, 1441-1449, Torres et al., Plant cellTissue and Organic Culture. 1993, 34: 3, 279-285, Dinant et al.,Molecular Breeding. 1997, 3: 1, 75-86. A. tumefaciens and A. rhizogenesare plant pathogenic soil bacteria which genetically transform plantcells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,respectively, carry genes responsible for genetic transformation of theplant. See, for example, Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991).Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are provided by Gruber et al.,supra, Miki et al., supra, and Moloney et al., Plant Cell Reports 8:238(1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer

Several methods of plant transformation collectively referred to asdirect gene transfer have been developed as an alternative toAgrobacterium-mediated transformation. A generally applicable method ofplant transformation is microprojectile-mediated transformation whereinDNA is carried on the surface of microprojectiles measuring 1 to 4 μm.The expression vector is introduced into plant tissues with a biolisticdevice that accelerates the microprojectiles to speeds of 300 to 600 m/swhich is sufficient to penetrate plant cell walls and membranes.Russell, D. R., et al. Pl. Cell. Rep. 12(3, January), 165-169 (1993),Aragao, F. J. L., et al. Plant Mol. Biol. 20(2, October), 357-359(1992), Aragao, F. J. L., et al. Pl. Cell. Rep. 12(9, July), 483-490(1993). Aragao, Theor. Appl. Genet. 93: 142-150 (1996), Kim, J.;Minamikawa, T. Plant Science 117: 131-138 (1996), Sanford et al., Part.Sci. Technol. 5:27 (1987), Sanford, J. C., Trends Biotech. 6:299 (1988),Klein et al., Bio/Technology 6:559-563 (1988), Sanford, J. C., PhysiolPlant 7:206 (1990), Klein et al., Biotechnology 10:268 (1992).

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively,liposome or spheroplast fusion has been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂) precipitation, polyvinyl alcohol orpoly-L-ornithine has also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Saker, M.; Kuhne, T. Biologia Plantarum 40(4): 507-514(1997/98), Donn et al., In Abstracts of VIIth International Congress onPlant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990); D'Halluin etal., Plant Cell 4:1495-1505 (1992) and Spencer et al., Plant Mol. Biol.24:51-61 (1994). See also Chupean et al., Biotechnology. 1989, 7: 5,503-508.

Following transformation of Cannabis target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic line. The transgenic line could then be crossed,with another (non-transformed or transformed) line, in order to producea new transgenic Cannabis line. Alternatively, a genetic trait that hasbeen engineered into a particular Cannabis cultivar using the foregoingtransformation techniques could be moved into another line usingtraditional backcrossing techniques that are well known in the plantbreeding arts. For example, a backcrossing approach could be used tomove an engineered trait from a public, non-elite inbred line into anelite inbred line, or from an inbred line containing a foreign gene inits genome into an inbred line or lines which do not contain that gene.As used herein, “crossing” can refer to a simple X by Y cross, or theprocess of backcrossing, depending on the context.

Gene Conversions

When the term Cannabis plant, cultivar or Cannabis line is used in thecontext of the present invention, this also includes any geneconversions of that line. The term gene converted plant as used hereinrefers to those Cannabis plants which are developed by a plant breedingtechnique called backcrossing wherein essentially all of the desiredmorphological and physiological characteristics of a cultivar arerecovered in addition to the gene transferred into the line via thebackcrossing technique. Backcrossing methods can be used with thepresent invention to improve or introduce a characteristic into theline. The term backcrossing as used herein refers to the repeatedcrossing of a hybrid progeny back to one of the parental Cannabis plantsfor that line. The parental Cannabis plant that contributes the gene forthe desired characteristic is termed the nonrecurrent or donor parent.This terminology refers to the fact that the nonrecurrent parent is usedone time in the backcross protocol and therefore does not recur. Theparental Cannabis plant to which the gene or genes from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Poehlman & Sleper,1994; Fehr, 1987). In a typical backcross protocol, the originalcultivar of interest (recurrent parent) is crossed to a second line(nonrecurrent parent) that carries the single gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until aCannabis plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred gene from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute traits or characteristics in the original line.To accomplish this, a gene or genes of the recurrent cultivar aremodified or substituted with the desired gene or genes from thenonrecurrent parent, while retaining essentially all of the rest of thedesired genetic, and therefore the desired physiological andmorphological, constitution of the original line. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross. One of the major purposes is to add some commerciallydesirable, agronomically important trait or traits to the plant. Theexact backcrossing protocol will depend on the characteristics or traitsbeing altered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

Many gene traits have been identified that are not regularly selectedfor in the development of a new line but that can be improved bybackcrossing techniques. Gene traits may or may not be transgenic,examples of these traits include but are not limited to, herbicideresistance, resistance for bacterial, fungal, or viral disease, insectresistance, enhanced nutritional quality, industrial usage, yieldstability, yield enhancement, male sterility, modified fatty acidmetabolism, and modified carbohydrate metabolism. These genes aregenerally inherited through the nucleus. Several of these gene traitsare described in U.S. Pat. Nos. 5,777,196; 5,948,957 and 5,969,212, thedisclosures of which are specifically hereby incorporated by reference.

Tissue Culture

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of Cannabis andregeneration of plants therefrom is well known and widely published. Forexample, reference may be had to Teng et al., HortScience. 1992, 27: 9,1030-1032 Teng et al., HortScience. 1993, 28: 6, 669-1671, Zhang et al.,Journal of Genetics and Breeding. 1992, 46: 3, 287-290, Webb et al.,Plant Cell Tissue and Organ Culture. 1994, 38: 1, 77-79, Curtis et al.,Journal of Experimental Botany. 1994, 45: 279, 1441-1449, Nagata et al.,Journal for the American Society for Horticultural Science. 2000, 125:6, 669-672, and Ibrahim et al., Plant Cell, Tissue and Organ Culture.(1992), 28(2): 139-145. It is clear from the literature that the stateof the art is such that these methods of obtaining plants are routinelyused and have a very high rate of success. Thus, another aspect of thisinvention is to provide cells which upon growth and differentiationproduce Cannabis plants having the physiological and morphologicalcharacteristics of cultivar NWG331 or NWG452.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, meristematic cells, andplant cells that can generate tissue culture that are intact in plantsor parts of plants, such as leaves, pollen, embryos, roots, root tips,anthers, pistils, flowers, seeds, petioles, suckers and the like. Meansfor preparing and maintaining plant tissue culture are well known in theart. By way of example, a tissue culture comprising organs has been usedto produce regenerated plants. U.S. Pat. Nos. 5,959,185; 5,973,234 and5,977,445 describe certain techniques, the disclosures of which areincorporated herein by reference.

Additional Breeding Methods

This invention also is directed to methods for producing a Cannabisplant by crossing a first parent Cannabis plant with a second parentCannabis plant wherein the first or second parent Cannabis plant is aCannabis plant of cultivar NWG331 or NWG452. Further, both first andsecond parent Cannabis plants can come from hemp Cannabis cultivars ofthe invention. Thus, any such methods using hemp Cannabis cultivars ofthe invention are part of this invention: selfing, backcrosses, hybridproduction, crosses to populations, and the like. All plants producedusing hemp Cannabis cultivars of the invention as at least one parentare within the scope of this invention, including those developed fromcultivars derived from hemp Cannabis cultivars of the invention.Advantageously, this Cannabis cultivar could be used in crosses withother, different, Cannabis plants to produce the first generation (F₁)Cannabis hybrid seeds and plants with superior characteristics. Thecultivar of the invention can also be used for transformation whereexogenous genes are introduced and expressed by the cultivar of theinvention. Genetic variants created either through traditional breedingmethods using hemp Cannabis cultivars of the invention or throughtransformation of cultivar NWG331 or NWG452 by any of a number ofprotocols known to those of skill in the art are intended to be withinthe scope of this invention.

The following describes breeding methods that may be used with hempCannabis cultivars of the invention in the development of furtherCannabis plants. One such embodiment is a method for developing cultivarNWG331 or NWG452 progeny Cannabis plants in a Cannabis plant breedingprogram comprising: obtaining the Cannabis plant, or a part thereof, ofcultivar NWG331 or NWG452, utilizing said plant or plant part as asource of breeding material, and selecting a hemp Cannabis cultivars ofthe invention progeny plant with molecular markers in common withcultivar NWG331 or NWG452 and/or with morphological and/or physiologicalcharacteristics selected from the characteristics listed in Table 1.Breeding steps that may be used in the Cannabis plant breeding programinclude pedigree breeding, backcrossing, mutation breeding, andrecurrent selection. In conjunction with these steps, techniques such asRFLP-enhanced selection, genetic marker enhanced selection (for exampleSSR markers) and the making of double haploids may be utilized.

Another method which may be used involves producing a population of hempCannabis cultivars of the invention-progeny Cannabis plants, comprisingcrossing cultivar NWG331 or NWG452 with another Cannabis plant, therebyproducing a population of Cannabis plants, which, on average, derive 50%of their alleles from hemp Cannabis cultivars of the invention. A plantof this population may be selected and repeatedly selfed or sibbed witha Cannabis cultivar resulting from these successive filial generations.One embodiment of this invention is the Cannabis cultivar produced bythis method and that has obtained at least 50% of its alleles from hempCannabis cultivars of the invention.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr and Walt, Principles of CultivarDevelopment, p 261-286 (1987). Thus the invention includes hemp Cannabiscultivars of the invention progeny Cannabis plants comprising acombination of at least two cultivar NWG331 or NWG452 traits selectedfrom the group consisting of those listed in Table 1 or the cultivarNWG331 or NWG452 combination of traits listed above, so that saidprogeny Cannabis plant is not significantly different for said traitsthan hemp Cannabis cultivars of the invention as determined at the 5%significance level when grown in the same environmental conditions.Using techniques described herein, molecular markers may be used toidentify said progeny plant as a hemp Cannabis cultivars of theinvention progeny plant. Mean trait values may be used to determinewhether trait differences are significant, and preferably the traits aremeasured on plants grown under the same environmental conditions. Oncesuch a variety is developed its value is substantial since it isimportant to advance the germplasm base as a whole in order to maintainor improve traits such as yield, disease resistance, pest resistance,and plant performance in extreme environmental conditions.

Progeny of hemp Cannabis cultivars of the invention may also becharacterized through their filial relationship with hemp Cannabiscultivars of the invention, as for example, being within a certainnumber of breeding crosses of hemp Cannabis cultivars of the invention.A breeding cross is a cross made to introduce new genetics into theprogeny, and is distinguished from a cross, such as a self or a sibcross, made to select among existing genetic alleles. The lower thenumber of breeding crosses in the pedigree, the closer the relationshipbetween hemp Cannabis cultivars of the invention and its progeny. Forexample, progeny produced by the methods described herein may be within1, 2, 3, 4 or 5 breeding crosses of hemp Cannabis cultivars of theinvention.

The foregoing invention has been described in detail by way ofillustration and example for purposes of clarity and understanding.However, it will be obvious that certain changes and modifications suchas single gene modifications and mutations, somoclonal variants, variantindividuals selected from large populations of the plants of the instantvariety and the like may be practiced within the scope of the invention,as limited only by the scope of the appended claims.

DEPOSITS

Applicant(s) made a deposit of at least 2500 seeds of Hemp Cannabiscultivars NWG331 and NWG452 with an International Depositary Authorityas established under the Budapest Treaty according to 37 CFR1.803(a)(1), at the National Collections of Industrial, Food and MarineBacteria Ltd. (NCIMB) in Aberdeen Scotland, Accession No. NCIMB 43290and NCIMB 43280. The NWG331 and NWG452 seeds deposited therewith on Nov.23, 2018 and Nov. 22, 2018, respectively, were taken from the depositmaintained by New West Genetics, PO Box 1662 Fort Collins, Colo. 80522since prior to the filing date of this application. Access to thisdeposit will be available during the pendency of the application to theCommissioner of Patents and Trademarks and persons determined by theCommissioner to be entitled thereto upon request. Upon issue of claims,the Applicant(s) will make available to the public, pursuant to 37 CFR1.808, a deposit of at least 2500 seeds of cultivar NWG331 and NWG452with an International Depositary Authority as established under theBudapest Treaty according to 37 CFR 1.803(a)(1), at the NationalCollections of Industrial, Food and Marine Bacteria Ltd. (NCIMB) inAberdeen Scotland.

This deposit will be maintained in the depository, which is a publicdepository, for a period of 30 years, or 5 years after the most recentrequest, or for the enforceable life of the patent, whichever is longer,and will be replaced if it becomes nonviable during that period.Additionally, Applicants have or will satisfy all the requirements of 37C.F.R. §§ 1.801-1.809, including providing an indication of theviability of the sample. Applicants have no authority to waive anyrestrictions imposed by law on the transfer of biological material orits transportation in commerce. Applicants do not waive any infringementof their rights granted under this patent or under the Plant VarietyProtection Act (7 USC 2321 et seq.).

What is claimed is:
 1. A non-transgenic Cannabis cultivar with a THCcontent of 0.2% or less.
 2. The Cannabis cultivar of claim 1 whereinsaid cultivar has the cultivar of NWG331 or NWG452 as an ancestor. 3.Seed of Cannabis cultivar designated NWG331 or NWG452, wherein arepresentative sample of seed of said cultivar was deposited underAccession No. NCIMB 43290 or NCIMB
 43280. 4. A Cannabis plant, or a partthereof, produced by growing the seed of claim
 3. 5. A tissue culture ofcells produced from the plant of claim 4, wherein said cells of thetissue culture are produced from a plant part selected from the groupconsisting of embryo, meristematic cell, leaf, cotyledon, hypocotyl,stem, root, root tip, pistil, anther, flower, seed and pollen.
 6. Aprotoplast produced from the plant of claim
 4. 7. A protoplast producedfrom the tissue culture of claim
 5. 8. A Cannabis plant regenerated fromthe tissue culture of claim 5, wherein the plant has all of themorphological and physiological characteristics of cultivar NWG331 orNWG452, wherein a representative sample of seed was deposited underAccession No. NCIMB 43290 or NCIMB
 43280. 9. A method for producing ahybrid Cannabis seed, wherein the method comprises crossing the plant ofclaim 1 with a different Cannabis plant and harvesting the resultant F₁hybrid Cannabis seed.
 10. A hybrid Cannabis seed produced by the methodof claim
 9. 11. A hybrid Cannabis plant, or a part thereof, produced bygrowing said hybrid seed of claim
 10. 12. A method of producing a malesterile Cannabis plant wherein the method comprises transforming theCannabis plant of claim 1 with a nucleic acid molecule that confers malesterility.
 13. A male sterile Cannabis plant produced by the method ofclaim
 12. 14. A method for producing an herbicide resistant Cannabisplant wherein the method comprises transforming the Cannabis plant ofclaim 1 with a transgene, wherein the transgene confers resistance to anherbicide selected from the group consisting of imidazolinone,sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine andbenzonitrile.
 15. An herbicide resistant Cannabis plant produced by themethod of claim
 14. 16. A method of producing an insect resistantCannabis plant wherein the method comprises transforming the Cannabisplant of claim 1 with a transgene that confers insect resistance.
 17. Aninsect resistant Cannabis plant produced by the method of claim
 16. 18.The Cannabis plant of claim 17 wherein the transgene encodes a Bacillusthuringiensis endotoxin.
 19. A method of producing a disease resistantCannabis plant wherein the method comprises transforming the Cannabisplant of claim 1 with a transgene that confers disease resistance.
 20. Adisease resistant Cannabis plant produced by the method of claim
 19. 21.A method of producing a Cannabis plant with a value-added trait, whereinthe method comprises transforming the Cannabis plant of claim 1 with aheterologous nucleic acid sequence.
 22. A Cannabis plant with avalue-added trait produced by the method of claim
 21. 23. A method ofintroducing a desired trait into hemp Cannabis cultivars of theinvention wherein the method comprises: a) crossing a NWG331 or NWG452plant grown from NWG331 or NWG452 seed, wherein a representative sampleof seed was deposited under Accession No. NCIMB 43290 or NCIMB 43280,with a plant of another Cannabis cultivar that comprises a desired traitto produce F₁ progeny plants, wherein the desired trait is selected fromthe group consisting of altered terpene or cannabinoid composition, malesterility, herbicide resistance, insect resistance, and resistance tobacterial disease, fungal disease, or viral disease; b) selecting one ormore progeny plants that have the desired trait to produce selectedprogeny plants; c) crossing the selected progeny plants with the NWG331or NWG452 plants to produce backcross progeny plants; d) selecting forbackcross progeny plants that have the desired trait and all of thecharacteristics of hemp Cannabis cultivars of the invention listed inTable 1 or Table 3 to produce selected backcross progeny plants; and e)repeating steps (c) and (d) three or more times in succession to produceselected fourth or higher backcross progeny plants that comprise thedesired trait and all of the characteristics of hemp Cannabis cultivarsof the invention listed in Table 1 or Table
 3. 24. A Cannabis plantproduced by the method of claim 23, wherein the plant has the desiredtrait and all of the characteristics of hemp Cannabis cultivars of theinvention listed in Table 1 or Table
 3. 25. The Cannabis plant of claim24, wherein the desired trait is herbicide resistance and the resistanceis conferred to an herbicide selected from the group consisting ofimidazolinone, sulfonylurea, glyphosate, glufosinate,L-phosphinothricin, triazine and benzonitrile.
 26. The Cannabis plant ofclaim 24, wherein the desired trait is insect resistance and the insectresistance is conferred by a transgene encoding a Bacillus thuringiensisendotoxin.
 27. The Cannabis plant of claim 24, wherein the desired traitis male sterility and the trait is conferred by a nucleic acid molecule.28. A method of conferring aroma, flavoring, or desired health benefitsto a beverage comprising; preparing said beverage with the Cannabisplant of claim 1, or parts thereof, or compositions purified therefrom.29. The method of claim 28 wherein said beverage is beer, wine, cider,distilled spirit, hard soda, soft drink, juice, water, or flavoredwater.
 30. A method of preparing cannabinoid isolates or isolateformulations, wherein the method comprises: harvesting flower tissuefrom the plant of claim 1; and extracting cannabinoids from the flowertissue.
 31. A cannabis product produced or purified from the cannabisplant, or part thereof, of claim
 1. 32. The cannabis product of claim31, wherein the product is produced or purified from seed.
 33. Thecannabis product of claim 31, wherein the product is a textile, abuilding material, a food or beverage, a personal hygiene product, apharmaceutical or medicinal product, an industrial product, or an animalfeed.
 34. A method of producing a Cannabis plant with a modified terpeneor cannabinoid profile wherein the method comprises geneticallymodifying the Cannabis plant of claim 1 with a nucleic acid moleculethat modifies the terpene or cannabinoid profile.
 35. A Cannabis plantwith a modified terpene or cannabinoid profile produced by the method ofclaim
 34. 36. A method of producing a Cannabis plant with a THC contentof 0.2% or less comprising the steps of: (a) crossing the plant of claim1 with a second Cannabis plant to produce a progeny plant; (b) crossingthe progeny plant of step (a) with itself or the second Cannabis plantin step (a) to produce a seed; (c) growing a progeny plant of asubsequent generation from the seed produced in step (b); (d) crossingthe progeny plant of a subsequent generation of step (c) with itself orthe second Cannabis plant in step (a) to produce a Cannabis plant with aTHC content of 0.2% or less.
 37. A method for developing a Cannabisplant in a Cannabis plant breeding program, comprising applying plantbreeding techniques comprising recurrent selection, backcrossing,pedigree breeding, marker enhanced selection, mutation breeding, orgenetic modification to the Cannabis plant of claim 1, or its parts, todevelop of a Cannabis plant with a THC content of 0.2% or less.